DESCRIPTION (Investigator's Abstract): Our goal is to elucidate the factors that regulate intracellular calcium in mammalian neurons (including nerve terminals) and astrocytes, and to determine how they influence physiological and pathophysiological processes. The Na/Ca exchange transport system, present in the plasmalemma of neurons and astrocytes, has the capacity to move large amounts of Ca2+ very quickly; it appears to play a dominant role in the extrusion of Ca2+ from these cells following activation. Therefore, much of the focus of this project is on the role played by the plasmalemmal Na/Ca exchanger in the brain: 1) its contribution to the control of intracellular free Ca2+ and stored Ca2+, 2) its influence on the responsiveness of neurons and astrocytes to depolarization, transmitters, and excitotoxins, and 3) its relationship to neuronal cell injury and death. To this end, the detailed kinetic properties (maximal flux rates, ion affinities, etc.) of the Na/Ca exchanger will be determined in rat forebrain nerve terminals (synaptosomes). These data will be compared to similar (but limited) data that will be obtained from bovine neurohypophyseal synaptosomes. Intra-terminal Ca2+ homeostasis in bovine neurohypophyseal synaptosomes will then be studied with the fluorescent indicators, fura-2 (for Ca2+) and SBFI (for Na+ measurements, related to Na/Ca exchange), using digital imaging methods. In parallel, fura-2 and SBFI will be used with digital imaging methods to examine the role of Na/Ca exchange in cultured rat striatal neurons and astrocytes to determine the role of the exchanger in regulating the responsiveness of these cells to neurotransmitters, hypoxia and excitotoxins. Finally, the investigators will employ a contribution of immunocytochemical methods with a polyclonal antibody to the exchanger, and in situ hybridization methods with a specific probe for the rat brain exchanger, to determine its distribution in the brain at various times during development and in the adult. The results of these studies should help us to understand the pathogenesis of (and perhaps develop new therapeutic strategies for) the altered cell Ca2+ metabolism that contributes to neuronal cell injury and cell death in a wide variety of neuronal diseases.